Biological systems are very complex and robust. One method for understanding complexity is to break the systems in smaller parts, characterize the parts and on the basis of this predict the behavior of complex part. Broad area of my project is to explore the behavior of complex systems by breaking it in small parts and characterizing them, using synthetic networks.

1.Reverse Engineering LuxI/LuxR Quorum-Sensing Systems.Recent advances in the study of bacterial gene expression have discovered that many bacteria employ a dedicated inter- and/or intra-species communication system. This bacterial decision-making system enables a given species to sense, integrate and process information from its surroundings, communicate with each other, and monitor its own population density and, as a response, activate or repress specific gene expression. This bacterial cell-density-dependent communication system is known as quorum sensing. The regulation of bioluminescence in Vibrio fischeri was the first quorum sensing phenomenon to be studied and V. fischeri has subsequently become a model for studies of the mechanism of quorum sensing. The autoinducer in V. fischeri is identified as N- (3-oxohexanoyl)-homoserine lactone. In V. fischeri, LuxR which is a transcriptional factor (also known as autoinducer receptor protein) and the LuxI, which is the AHL synthase proteins as well as a proposed LuxR binding site lux box (a 20 bp inverted repeat situated within the lux regulon) are necessary for the activation of quorum sensing related gene expression. LuxR and LuxI proteins are constitutively expressed at low cell densities. AHL accumulates in proportion to the increasing bacterial population. When the AHL molecules reach at a certain threshold concentration, binding to receptor molecules is promoted and the activated LuxR–AHL complex forms multimers which enables the LuxR protein to bind with the lux box and act as a transcriptional activator, thus enhancing the expression of the target genes of the QS systems. Since, luxI is located within this operon, more AHL is produced, more AHL: LuxR binding occurs and increases transcriptional activation of the luxI, thereby generating a positive feedback regulatory loop in which AHL controls its own synthesis. We reverse engineered V. fischeri LuxI/LuxR quorum sensing system and redesigned it into three synthetic quorum sensing systems. We predicted the responses of all three V. fischeri synthetic quorum sensing systems, also called closed loops here, from the characteristics of an open loop and found that closed loop responses justify the open loop predictions. We used three transcriptional regulators (LacI, TetR and LuxI/LuxR module) and two fluorescent reporter proteins (CFP and YFP), to design all of our networks. We made predictions of two closed loops i.e. LuxR and LuxI closed loop and found that LuxR closed loop is monostable while LuxI closed loop is bistable. We tested these predictions by doing experiments on closed loop themselves as well as through computational modeling and found that predictions match Close loop responses qualitatively and quantitatively. Our work confirms experimentally that it is possible to predict closed loop responses from open loop characteristic curves (as demonstrated theoretically by Angeli et al. PNAS 2004)

2.Development of multi-cell oscillatory genetic circuitOne of the central focuses in post-genomic research is to understand how biological phenomena arise from the interactions between genes and proteins. The molecular mechanism by which oscillation in gene expression are sustained in organisms, have been widely studied by biologists using organisms from cyanobacteria to human. But regulation of these natural clocks is very complex and it is very difficult to find out parameters operating these clocks. Due to development in post-genomic research it is feasible to design synthetic genetic circuits, mimicking the properties of natural clocks, hence will provide a tool to understand design principle of natural clocks.Biologists have developed several circadian clocks. These clocks are not as robust as natural, but at least disclose the information of key parameters regulating these circuits. Synthetic oscillatory circuits include repressilator, metabolic gene oscillator, and tunable synthetic mammalian oscillator In present study, we are developing multi-cell oscillatory genetic circuit, consisting of a positive and a negative feedback.